Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

(1)

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

TYROSINE KINASE-

LINKED RECEPTORS PART 2

GROWTH FACTORS

Tímea Berki and Ferenc Boldizsár Signal transduction

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(3)

Growth factors (GFs)

• Small molecular weight soluble mediators

• They control:

1 Proliferation 2 Survival

3 Metabolism

4 Tissue differentiation

• Important implication in tumors

• Cytokines – growth factors

(4)

Growth factors (GFs) – History

How to propagate cells under in vitro conditions (turning of the 19 ^th -20 ^th century)?

• Rous: experiments with chicken tumor (sarcoma) cells (RSV)

• Carrel: In simple buffered salt solution the cells did not proliferate – trials with diluted plasma/serum

• Temin and Dulbecco: precise requirements for tissue culturing:

– Reduced serum need of tumor cells – enhanced capacity of tumor cells to respond to proliferation signals

– Serum supported cell growth rather than plasma – PDGF Rita Levi-Montalcini, Stanley Cohen – NGF and EGF

Transplantation of an actively growing mouse tumor into chicken induced

the great amplification of nerve fibres into the tumor mass

(5)

Growth factors

Factor Principal source Primal activity Comments

PDGF Platelets, endothelial cells, placenta

Promotes proliferation of connective tissue, glial and smooth muscle cells

Two differentprotein chains from 3 distinct dimer forms; AA, AB and BB EGF Submaxillary gland, Brunners

gland

Promotes proliferation of mesenchymal, glial and epithelial cells

TGF-α Common in transformed cells May be important for normal wound

healing Related to EGF

FGF Wide range of cells; protein is associated with the ECM

Promotes proliferation of many cells;

inhibits some stem cells; induces mesoderm to form in early embrios

At least 18 family members, 5 distinct receptors

NGF

Mast cells, eosinophils, bone marrow stromal cells, keratinocytes

Promotes neurite outgrowth and neural cell survival

Member of a family of proteins termed neutrophins that promote proliferation and survival of neurons; neutrophin receptors are a class of related proteins first identified as proto-oncogenes? TrkA („trackA”), TrkB, TrkC

Erythropoietin Kidney Promotes proliferation of erythrocytes

TGF-β Activated Th1 cells (T-helper) and natural killer (NK) cells

Anti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits

macrophage and lymphocyte proliferation

At least 100 different family members

IGF-1 Primarily liver Promotes proliferation of many cell types Related to IGF-2 and proinsulin, also called somatomedin C

IGF-2 Variety of cells Promotes proliferation of many cell types

primarily of fetal origin Related to IGF-1 and proinsulin

(6)

Receptors with TK activity

• Receptor Tyrosine Kinases (RTK)

e.g. PDGF, insulin, EGF, VEGF and FGF receptors

• Tyrosine-Kinase Associated Receptors

Receptors that associate with proteins that have tyrosine kinase activity (Cytokine Receptors)

• Receptor Tyrosine Phosphatases

e.g. CD45 protein of T cells and macrophages

(7)

“Complete” and “incomplete” receptor tyrosine kinase

“ Complete ” receptor tyrosine kinase “ Incomplete ” receptor

Cytoplasmic non-receptor tyrosine kinase Adaptor

Signal Signal

(8)

Receptor tyrosine kinase (RTK) families

• 90 unique Tyr kinases in the human genome, 58 are RTKs

• Growth factor, cytokine and hormone receptors

• Classes:

I EGFR family (ErbB) X LTK family II Insulin rec. family XI TIE family III PDGF family XII ROR family IV FGF family XIII DDR family V VEGF family XIV RET family VI HGF family (c-Met) XV KLG family VII Trk family XVI RYK family VIII Eph family XVII MuSK family

IX AXL family

(9)

Kinase-phosphatase balance

Phosphorylase kinase (ser/thr kinase)

PP1c (ser/thr phosphatase) Phosphorylase b

Phosphorylase b

Phosphorylase a P Phosphorylase a P

Inactive Active

P

ATP ADP

CD45 (tyr phosphatase)

Csk (tyr kinase)

ADP ATP

Inactive _p56Lck

P Y505

Y394

Primed _p56Lck

Y505

Y394

Active_p56Lck

P Y394 P

(10)

Fibronectin III Leucine-rich Cysteine-rich

Acid-box Kinase

IG-like

VEGFR1 VEGFR2 VEGFR3 PDGFR

PDGFRβ CSF1R Kit Kit2

Ryk Torso

EGFR ErbB2 ErbB3 ErbB4

Met Ron Sea

TrkA TrkB TrkC INSR

IGF1R IRR

Axl Mer Sky

Eph Eck Eek Erk Elk Ehk1 Ehk2 Sek Hek Hek11 Cek-9 Myk-1 Myk-2

Ros FGFR1

FGFR2 FGFR3 FGFR4

Tie Tie2

DDR Ret Ror1 Torpedo

Ror2 Ltk Alk

EGF-like Cadherin

Factor VIII-like

Glicyne-rich Kringle C1r-like

Growth factor receptors

(11)

Receptor-like PTPs (21)

CD45 (RC) R1/R6

PTP

(RM) PTP

(RK) PTP

(RT) PTP

(RU) R2B

LAR (RF) PYP

(RS) PTP

(RD) R2A

PTP

(RA) PYP

(RE) R4

PTP

(RG) PYP

(RZ1) R5

PTPβ (RB) DEP1

(RJ) SAP1

(RH) GLEPP1

(RO) PTPS31

(RP) R3

PCPTP1

(RR) STEP

(N5) R7

IA2 (RN) IA2β (RN2)

R8

RGDS motif

Proline-rich MAM domain

SEC14 domain Fibronectin III

FERM domain IG-like

BRO-1 homology Glycosylated

PDZ domain Cadherine-like

Histidine domain Carbonic anhydrase-like

Kinase-interacting domain PTP domain

Src homology 2 PTP pseudo-phosphatase

domain

(12)

Nontransmembrane PTPs (17)

HDPTP (N23)

NT8 MEG2

(N9) NT3

HePTP (N7) PTPH1 (N3) MEG1

(N14) NT5

SHP1 (N6) SHP2 (N11) NT2

PTPBAS (N13)

NT7

PTPD1 (N21) PTPD2β

(142) NT6 PTP1B

(N1) TCPTPβ

(N2) NT1

BDP1 (N18) PTP-PEST

(N12) LYP (N220)

NT4

PTPTyP (N20)

NT9

RGDS motif

Proline-rich MAM domain

SEC14 domain Fibronectin III

FERM domain IG-like

BRO-1 homology Glycosylated

PDZ domain Cadherine-like

Histidine domain Carbonic anhydrase-like

Kinase-interacting domain PTP domain

Src homology 2 PTP pseudo-phosphatase

domain

(13)

Growth factor receptors and tyrosine phosphorylation

p120 Ras-Gap PLC

Phosphotyrosine

Y559 Y581 Y716 Y741 Y751 Y771 Y1009 Y1021

Phosphotyrosine

Y992 Y1045 Y1068 Y1086 Y1148 Y1173

PDGFR

P P P

P P

P P P

P P

EGFR

P P P P

P

P P P

P P

Shc SLP7

6 Abl

Cbl PI3K

IRS-1

GRB2 SHP- SRC

1 STAT1 Crk

Nck

(14)

Receptor tyrosine kinase (RTK) signaling

Proliferation Survival Migration Cell cycle progression Transcription

RTK

Ligand

P

P P P P

P P P Dimerization

Src

SOS GRB2

Ras

Raf Erk PKC

PLC

STAT JAK

Akt PI3K PDK1

(15)

GF receptor signaling pathways I

Targets

PIP

₂

PIP

₃

Targets Targets

Akt PDK1

PIP

₃

SOS Ras

Targets

Erk

Targets GRB2

GRB2 Shp2

Shp2

GRB2 GRB2

GRB2 PI3K RTK

Ligand

P P

P P P

P

P P

P

Plasma membrane

P

P P P P

P P P

(16)

GF receptor signaling pathways II

RTK Ligand

DAG IP

₃

PIP

₂

PKC

Ca²⁺

Cbl

P

PLC PLC C2

PH SH2 SH2 SH3

Plasma membrane

Cytoplasm

P

P P P P

P P P

(17)

Overview of EGF signaling

EGFR

JNK SOS

Ras GRB2

C-Fos Raf

MAPKK

AP1

MAPK PAK1

Nck

GRB2

Gab1

Shp2

WASP Src

Shc

Bad FKHR

CREB

RSK2 p53 Jun

MAPK p38 JNK

Cdc42 /Rac Vav2

EGF

Cytoskeleton Cell cycle

Apoptosis

STAT1 STAT3

Target genes

ADAM

HB-EGF PTP

Rac H₂O₂

NADPH

synthesis Gab1

PI3K

PIP₃ E₂Ub

Targets

DOK

Akt PDK1

Cbl

MKK2 MKK4

MEKK MEKK4

Rac FAK

CAS

Paxillin Src

Targets Ca²⁺

PKC

DAG PLC

IP₃

MAPK

Ras GAP +

- -

- +

(18)

General characteristics of GF signaling

Diverse input signals (Multiple RTKs)

Conserved core processes

Diverse ouput events (transcriptional responses,

cytokeletal changes, etc) System control

+

- +

+ - Input layer

Output layer

(19)

Different GF receptors use the same signaling pathways

PDGF-C

Cell survival Proliferation Apoptosis resistance Metastasis Angiogenesis

P

P P P P

P P P

P

P P P P

P P P

P

P P P P

P P P

P

P P P P

P P P

P

P P P P

P P P

EGFR Her2 Her3 Her4 VEGFR1 VEGFR2 VEGFR3 PDGFR-a PDGFR-b c-kit

EGF TGF

β-cellulin Amphiregulin

HB-EGF

No specific

ligands Heregulins

β-cellulin NRG2

NRG3 VEGF-B VEGF-A VEGF-C VEGF-D PDGF-A

PDGF-B

PDGF-D SCF

SOS

GRB2 Ras Rac CDC42 Rho

MEK1/2

Erk Raf ERK pathway

MKK4/7

JNK MEKK JNK pathway

MKK3/6

p38 Tak p38 pathway

Akt

mTor PI3K

Everolimus Imatinib Trastuzumab Leflunomide Lapatinib Gefitinib Erlotinib Panitumumab Sorafenib

Cetuximab Bevacizumab Vandetanib Sunitinib Enzastaurin Pazopanib Motesanib Midostaurin Temsirolimus Sirolimus

P

P P P P

P P P

(20)

Natriuretic peptide signaling

↑NP degradation

↓cAMP?

↑ IP3?

↑ Vasorelaxation

↑ Diuresis, natriuresis

↓ Renin, aldosterone

↓ Cell proliferation

↓ Cardiac fibrosis

↑ Vasorelaxation

↓ Cell proliferation

↑ Long bone gowth Kinase homology domain

Plasma membrane Ligand binding domain Receptor

Hinge region

Guanylyl cyclase domain

Physiologic response Natriuretic peptide

NPR-C NPR-A

(GC-A)

NPR-B (GC-B)

ANP BNP CNP

cGMP

GTP GTP cGMP

P P P P P P

P P P P P

P P P P P P P P P P

P

Natriuretic peptide

Hormone bound

Active Desensitised

Kinase

Phosphatase ATP

ATP

cGMP GTP

P P

P

P P

P P Basal

ATP

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

TYROSINE KINASE-

LINKED RECEPTORS PART 2

GROWTH FACTORS

Tímea Berki and Ferenc Boldizsár Signal transduction

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

Growth factors (GFs)

• Small molecular weight soluble mediators

• They control:

1 Proliferation 2 Survival

3 Metabolism

4 Tissue differentiation

• Important implication in tumors

• Cytokines – growth factors

Growth factors (GFs) – History

How to propagate cells under in vitro conditions (turning of the 19 th -20 th century)?

• Rous: experiments with chicken tumor (sarcoma) cells (RSV)

• Carrel: In simple buffered salt solution the cells did not proliferate – trials with diluted plasma/serum

• Temin and Dulbecco: precise requirements for tissue culturing:

– Reduced serum need of tumor cells – enhanced capacity of tumor cells to respond to proliferation signals

– Serum supported cell growth rather than plasma – PDGF Rita Levi-Montalcini, Stanley Cohen – NGF and EGF

Transplantation of an actively growing mouse tumor into chicken induced

the great amplification of nerve fibres into the tumor mass

Growth factors

Receptors with TK activity

• Receptor Tyrosine Kinases (RTK)

e.g. PDGF, insulin, EGF, VEGF and FGF receptors

• Tyrosine-Kinase Associated Receptors

Receptors that associate with proteins that have tyrosine kinase activity (Cytokine Receptors)

• Receptor Tyrosine Phosphatases

e.g. CD45 protein of T cells and macrophages

“Complete” and “incomplete” receptor tyrosine kinase

“ Complete ” receptor tyrosine kinase “ Incomplete ” receptor

Cytoplasmic non-receptor tyrosine kinase Adaptor

Signal Signal

Receptor tyrosine kinase (RTK) families

• 90 unique Tyr kinases in the human genome, 58 are RTKs

• Growth factor, cytokine and hormone receptors

• Classes:

I EGFR family (ErbB) X LTK family II Insulin rec. family XI TIE family III PDGF family XII ROR family IV FGF family XIII DDR family V VEGF family XIV RET family VI HGF family (c-Met) XV KLG family VII Trk family XVI RYK family VIII Eph family XVII MuSK family

IX AXL family

Kinase-phosphatase balance

Growth factor receptors

Receptor-like PTPs (21)

Nontransmembrane PTPs (17)

Growth factor receptors and tyrosine phosphorylation

Phosphotyrosine

Phosphotyrosine

PDGFR

EGFR

Receptor tyrosine kinase (RTK) signaling

GF receptor signaling pathways I

Targets

PIP

PIP

Targets Targets

Akt PDK1

PIP

SOS Ras

Targets

Erk

Targets GRB2

GRB2 Shp2

Shp2

GRB2 GRB2

GRB2 PI3K RTK

Ligand

Plasma membrane

GF receptor signaling pathways II

RTK Ligand

DAG IP

PIP

PKC

Cbl

PLC PLC C2

How to propagate cells under in vitro conditions (turning of the 19 ^th -20 ^th century)?